Project Summary Protection and subsequent de-protection of centromeric cohesion during mitosis is essential for preventing chromosome missegregation and preserving genomic stability. This timing control of centromeric cohesion is defined by the inner-centromeric (the place between two sister centromeres) installment and removal of Shugoshin (Sgo1) proteins, but the underlying mechanisms that control the Sgo1 localization at inner centromeres are little known. The long-term goal is to understand the molecular mechanisms of chromosome segregation and their relationships with aneuploidy-driven processes and diseases. The objective of this proposal is to determine the essential mechanisms controlling the inner-centromere localization of Sgo1. We previously found that Bub1-dependent RNA polymerase II transcription is involved in installing Sgo1 at inner centromeres after initial kinetochore recruitment. Our preliminary data show that SET, a cellular PP2A inhibitor, can directly bind Sgo1 and inhibit the Sgo1-cohesin interaction. The central hypothesis is that Bub1-dependent actively-transcribing RNAP (RNA polymerase) II binds and delivers Sgo1 to inner centromeres at early mitosis, and that SET and/or PP1 (phosphatase 1) removes Sgo1 from inner centromeres at metaphase-to-anaphase transition. The rationale underlying this research is that the exploration of the regulation of centromeric cohesion is critical to understand its role in genome stability. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine the function and regulation of centromeric transcription in mitosis; and 2) Determine how centromeric cohesion is de-protected. Under the first aim, we will determine if Bub1-H2A-pT120 maintains the transcription-promoting epigenetic histone marks to facilitate transcription, and if elongating RNAP II binds and delivers Sgo1 to centromeric cohesin, thus enabling Sgo1 function. Under the second aim, we will determine if SET and/or PP1 removes Sgo1 from inner centromeres at metaphase-to-anaphase transition by disrupting the Sgo1-cohein interaction. The proposed research is innovative, in the application?s opinion, because it departs the status quo by revealing totally novel and important mechanisms that determine the timing control of centromeric cohesion. This research is also significant, because it is expected to vertically advance and expand understanding of the molecular mechanisms that are essential for proper chromosome segregation and prevent aneuploidy. Ultimately, such knowledge will help identify potential targets for future development of new anti-cancer therapy.